The removed brain was then rapidly immobilized in 4% paraformaldehyde in PBS, embedded in paraffin, sectioned, and stained with H&E according to standard protocols. The H&E‐stained brain section images were taken by an optical microscope (Olympus), which were further compared with a pH map to determine whether the glioma infiltrating boundary defined by CP2‐M matches the actual boundary indicated by the pathological result. The removed brain was made into frozen sections, and the CP2‐M signal and bright‐field images were taken by confocal microscopy to verify probe entry into the brain. The altered frozen sections were subjected to immunofluorescence staining to verify the co‐localization of CP2‐M with CD206 in vivo, antibody information: CD206 (proteintech, 60143‐1, 1:50).
Cd206
Manufactured by Proteintech
Sourced in China, United States, United Kingdom
CD206 is a protein expressed on the cell surface of macrophages and other antigen-presenting cells. It functions as a receptor that mediates the endocytosis of glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
F4 80, by Proteintech (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
F4 80, by Cell Signaling Technology (4 mentions)
β actin, by Proteintech (3 mentions)
Tgf β, by Proteintech (3 mentions)
Cd163, by Proteintech (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Gapdh, by Proteintech (2 mentions)
Bcl 2, by Proteintech (2 mentions)
F4 80, by Abcam (2 mentions)
Streptozotocin (stz), by Merck Group (2 mentions)
α sma, by Proteintech (2 mentions)
F4 80, by Santa Cruz Biotechnology (2 mentions)
Anti cd86 apc, by BD (2 mentions)
Anti f4 80 fitc, by BD (2 mentions)
Anti cd206 pe, by BD (2 mentions)
If staining of cd86, by ABclonal (2 mentions)
Vascular endothelial growth factor (vegf), by Proteintech (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
61 protocols using cd206
1
Histological Validation of Glioma Imaging Probe
2
Western Blot Antibody Validation
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3
Immunohistochemical Validation of EWS Biomarkers
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To further verify the accuracy of validating these three genes, six pairs of paraffin‐embedded EWS and adjacent tissues were collected for immunohistochemical analysis. This study was approved by the Institutional Review Board of Xijing Hospital, Fourth Military Medical University, and all patients signed informed consents. All tissue slices were subjected to a series of steps such as dewaxing, antigen retrieval, blocking, primary antibody incubation, and secondary antibody incubation, including CTSD (Proteintech, 21327‐1‐AP), SIRPA (Affinity, AF0253), FN1 (Proteintech, 15613‐1‐AP), CD8 (Proteintech, 66868‐1‐lg), CD206 (Proteintech, 60143‐1‐lg), CD66b (Abcam, ab300122), and CD57 (Servicebio, GB113461). Finally, the slices were subjected to DAB kit (CWBIO, CW2035S) color rendering and hematoxylin redye. The microscopy was used to observe protein expression for each stained section, and the positive rate of each immunohistochemically stained section was calculated using Image J software.
4
Cerebral Cortex Protein Expression Analysis
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The injured side cerebral cortex samples of rats in sham, SAH and EA groups on 1 day were homogenized in 10 vol of ice-cold RIPA (Beyotime, China) containing protease inhibitor cocktail (Roche, Indianapolis, USA). Prepared protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were probed overnight at 4 °Cwith the following primary antibodies: p-STAT3 (1:500, Santa, USA), STAT3 (1:500, CST, USA), NOX2 (1:500, Proteintech, China), iNOS (1:500, Abcam, UK), CD206 (1:500, Proteintech, China), Cleaved casepase 3 (1:500, Proteintech, China), Bcl-2 (1:500, Proteintech, China), Bax (1:500, Proteintech, China), GAPDH (1:1000, Proteintech, China) and β-actin (1:1000, Proteintech, China) followed by incubation with secondary antibodies conjugated with horseradish peroxidase (1:10000, Beyotime, China). The bands were visualized with an ECL western blotting kit (Beyotime, China), and photographed by a chemiluminescence imaging system (Bio-Rad, USA). Protein quantification was performed using Image Lab software (Bio-Rad, USA).
5
Immunofluorescence Analysis of Cardiac Inflammation
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For immunofluorescence, after fixed in 4% paraformaldehyde overnight, the hearts of 3 days after MI surgery were immersed in 30% sucrose for several hours, embedded in OCT compound and snap‐frozen in liquid nitrogen, and stored at −80°C. Sections (5 μm) were incubated with 3% BSA in PBST with 0.1% Triton for 1 hour at room temperature. Then, sections were incubated with primary antibodies against iNOS (1:200; Abcam), Arg1 (1:50; ProteinTech Group), TNF‐α (1:200; Abcam), CD206 (1:100; ProteinTech Group) or F4/80 (1:200; Abcam) overnight at 4°C. After washing with PBST three times for 5 minutes each, the sections were incubated with appropriate secondary antibodies (Alexa Fluor 488 AffiniPure donkey anti‐rabbit or Alexa Fluor 594 AffiniPure donkey anti‐rat [1:400; Jackson ImmunoResearch]) for 2 hours at room temperature. After triple additional washing with PBST, the sections were sealed with DAPI Fluoromount‐GTM (Yeasen) to identify nuclei and prevent fluorescence quenching. Fluorescence microscopy images were caught with a laser scanning confocal microscope (LSM880; Zeiss). We used the number of iNOS and F4/80 double‐positive cells per F4/80 positive cells at the infarcted border zone to document the M1/M polarization and used the number of Arg1 and F4/80 double‐positive cells per F4/80 positive cells to document the M2/M polarization.
6
Macrophage Polarization Modulation Protocol
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Tetraethylorthosilicate (TEOS), Na2CO3, ethanol, methanol, ammoniumhydroxide (NH3·H2O), hexadecyl-trimethyl-ammoniumbromide (CTAB) and potassium permanganate (KMnO4) were purchased from SinopharmChemReagent Co., Ltd. (China). Metformin, lipopoly-saccharide (LPS) and Recombinant Murine IFN-γ were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Murine IL-4 was provided by PeproTech Biotechnology Co., Ltd. (Suzhou, China). Polyclonal antibodies CD47, CD80 and CD206 were obtained from Proteintech Group, Inc. (Wuhan, China). Coumarin-6 was ordered from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Mouse Tumor Necrosis Factor Alpha and Mouse IL-10 ELISA kit was provided by ABclonal Biotechnology Co., Ltd. (Wuhan, China). ELISA kits of Mouse iNOS and Mouse Arg-1 were obtained from Jonln Biotechnology Co., Ltd. (Shanghai, China). FITC-Anti-Mouse CD80 Antibody, FITC-Anti-Mouse CD206 and APC-Anti-Mouse CD206 Antibody were provided by Elabscience Biotechnology Co., Ltd. (Wuhan, China). β-Actin, AMPKα (D63G4) Rabbit mAb and Phospho-AMPKα (Thr172) (40H9) Rabbit mAb were ordered from Cell Signaling Technology, Inc. (MA, USA). DSPE-PEG-M2pep was purchased from SunLipo NanoTech Co., Ltd. (Shanghai, China).
7
Protein Expression Analysis of Cell Lysates
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Cells were lysed in ice‐cold RIPA buffer containing protease inhibitor. The same amounts of protein were separated using an SDS‐PAGE gel and transferred to PVDF membranes (R1KB43643; Merck Co., Ltd.). The membranes were blocked in 5% skim milk (D8340; Solarbio Co., Ltd.) and then incubated overnight at 4°C with the primary antibodies for FoxO3a (66428‐1‐Ig; Proteintech Co., Ltd.), FoxO1 (18592‐1‐AP; Proteintech Co., Ltd.). Arg‐1 (66129‐1‐Ig; Proteintech Co., Ltd.), CD206 (60143‐1‐Ig; Proteintech Co., Ltd.), CD86 (13395‐1‐AP; Proteintech Co., Ltd.), GPX4 (A11243; ABclonnal Co., Ltd.), FTH1 (A19544; ABclonnal Co., Ltd.), HO‐1 (A19062; ABclonnal Co., Ltd.), LC3 (14600‐1‐AP; Proteintech Co., Ltd.), NCOA4 (A17330; ABclonnal Co., Ltd.), SLC7A11 (A2413; ABclonnal Co., Ltd.), β‐actin (HC201‐01; Transgen Co., Ltd.). Then subjected to HRP‐labelled secondary antibody for 1 h at room temperature. The reaction was visualised with chemiluminescent assay.
8
STZ-Induced Diabetic Kidney Fibrosis
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Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were from Gibco (Grand Island, NY). Streptozotocin (STZ) was obtained from Sigma Chemical (St. Louis, MO). Primary antibodies for immunohistochemistry and Western blot analyses included PTPN2 (ab180764; Abcam); P‐STAT1 (#9167; Cell Signaling); P‐STAT3 (#9145; Cell Signaling); Arginase I (16001‐1‐AP; Proteintech Group Inc); Arginase II (14825‐1‐AP; Proteintech Group Inc); CD11c (17342‐1‐AP; Proteintech Group Inc); CD206 (18704‐1‐AP; Proteintech Group Inc); F4/80 (27044‐1‐AP; Proteintech Group Inc); CD3 (17617‐1‐AP; Proteintech Group Inc); fibronectin (ab2413; Abcam); Collagen I (#84336; Cell Signaling); Collagen IV (ab6586; Abcam); TGF‐β (#3711; Cell Signaling); PAI‐1 (ab125687; Abcam); α‐SMA (#19245; Cell Signaling); MCP‐1(#2029; Cell Signaling); TNF‐α (ab6671; Abcam); IL‐6 (ab208113; Abcam); ICAM‐1 (ab171123; Abcam); vascular endothelial growth factor (VEGF; 19003‐1‐AP; Proteintech Group Inc); CD31 (#77699; Cell Signaling) and β‐actin (ab8226; Abcam). ELISA kits for MCP‐1 were from ab208979, Abcam.
9
Western Blot Analysis of Macrophage Proteins
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Western blot was performed as previously described [19 (link)]. In brief, the protein extracted from the macrophages was degenerated, electrophoresed, and transferred to the polyvinylidene fluoride membrane (Millipore). Subsequently, the membrane was blocked using 5% non-fat milk and incubated with primary antibodies—rabbit anti-PFKFB3 (Abcam), iNOS (inducible nitric oxide synthase) (Proteintech), CD206, Jak1, P-Jak1, Jak2, P-Jak2, Jak3, P-Jak3, Stat 1, P-stat1 (Y701), P-stat1 (Y727) (all from CST technology), and mouse anti-β-actin (Abcam)—overnight at 4 °C. On the following day, membranes were washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratory) and detected with an ECL reagent (Yeasen, Shanghai, China) using the chemiluminescence system (Tanon, Shanghai, China). The grayscale of the detected bands was assessed by ImageJ software (National Institutes of Health), and the results were expressed as fold changes after normalizing to β-actin.
10
Protein Extraction and Western Blot
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Lysis buffer composed with RIPA, 1% phenylmethanesulfonyl fluoride and 1% phosphatase inhibitor were used to obtain the total protein and the protein concentration was detected by the BCA protein assay kit. Ten percent SDS-PAGE was used for protein separation, followed by transfer onto PVDF membranes. Five percent BSA was used to block the membranes for 1 hr, and then incubated with the appropriate primary antibodies overnight, at 4ºC. Antibodies used in this study included CD163 (1:1000, Abcam, Cambridge, UK), CD206 (1:1000, Santa, USA), STAT6 (1:1000, Proteintech, Chicago USA), p-STAT6 (1:1000, CST, Boston, USA) and β-catenin (1:1000, Proteintech, Chicago, USA).
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